WESTERN BLOT OF LARGE AND MINORITY PROTEIN - (Jan/27/2010 )
I'm trying to detect one 260kDa protein by common western blot method. Actually I use 6% (bis)acrylamide self-made gels, make eletrophoresis for 1h30min and finally transfer to a PVDF membrane by using semi dry method. Moreover, I've made the following variations:
1.- I've tried to run from 30ug till 350ug protein (in this case, I could only observe a miser smear...)
2.- The transfer time was ranged from 15min till 2hrs at 20v.
3.- My lysis buffer consists in TRITONX100. However, I could try NP40 or SDS buffer and sonication.
Since now I failed to find any protein. Do you know some variation which could increase my method's efficiency??
Large protein - long run and long transfer would be my best suggestion. I would run at 100-120 v for several hours and then wet transfer overnight at 15 V.
Adding some SDS to your lysis buffer and running buffers will help with the run and transfer.